The homeostatic balance between proliferation and apoptosis is essential for the intestinal epithelium to function as a physiological and structural barrier. Intestinal epithelial cells have a high rate of cell turnover accompanied by an equally high rate of apoptosis. This normal apoptosis is essential for the hierarchical organization of the intestinal epithelium and apoptotic epithelial cells have been detected both at the base of the crypt as well as the villus tips of the small and large intestine. Defects in apoptosis are associated with several gastrointestinal conditions including villus atrophy, epithelial hyperplasia, loss of normal absorptive function and increased risk of tumorigenesis. Surprisingly, there are few data available on apoptosis as an important mechanism of action in vivo in the gastrointestinal epithelium. There are very comprehensible data supporting links between actin and apoptosis in eukaryotic cells from studies using drugs that affect actin turnover. However, few studies have established a causal link between microfilament reorganization and cell survival. Villin is an actin regulatory protein expressed in the gastrointestinal epithelial cells of the small and large intestine as well as in exocrine glands associated with the GI tract. We recently determined, for the first time, that villin is an epithelial cell-specific anti-apoptotic protein. Further, our preliminary studies suggest that in the absence of villin, this function may be fulfilled by a related actin- binding protein of the villin family, gelsolin. Villin is also cleaved during the apoptotic cycle and we have previously reported that the cleaved NH2-terminal fragment of villin is pro-apoptotic. Using yeast two hybrid, biochemical and cellular approaches, we have recently identified and validated the novel interaction of villin with the mitochondrial protein, ATP synthase 2-subunit, which now allows us to investigate the cellular and molecular mechanism(s) of microfilament- induced regulation of mitochondrial morphology and function and epithelial cell survival by villin. The novelty of our observation will allow us to demonstrate for the first time a causal relationship between microfilament regulation and cell survival in eukaryotic cells. To achieve this, experiments will be conducted under three major specific aims: (i) to characterize the cellular and molecular mechanism(s) of villin's anti- and pro-apoptotic functions and to identify the physiologic relevance of full-length and cleaved villin fragments respectively, in the maintenance of gastrointestinal homeostasis;(ii) to characterize the interaction of villin with ATP synthase 2- subunit to examine the relevance of this complex in the maintenance of gastrointestinal homeostasis;(iii) to characterize the villin-gelsolin double knock out mice to establish the role of villin and its homologous protein gelsolin in the regulation of gastrointestinal homeostasis. Our studies are vital for the rational design of new and more effective therapies for major intestinal epithelial diseases such as inflammatory bowel disease and cancer. PUBLIC HEALTH RELEVANCE: The intestinal tract fulfils essential roles as a digestive and absorptive surface and constitutes the largest immune organ. In addition the intestine is the anatomical location of diseases of enormous social and economic impact, like the inflammatory bowel disease (affecting about 1 person in every 500 in USA), the colon carcinoma (the second most frequent malignancy in developed countries) and numerous infectious diseases. Apoptosis plays an important role in determining the architecture of intestinal epithelia and also a part of the stress response of intestinal epithelial cells to toxic stimuli. In disease pathogenesis, apoptosis can either be inappropriately excessive or deficient and both of these have been implicated in bacterial, viral and parasitic infections, ischemia-reperfusion injury, inflammatory bowel disease, celiac disease and colorectal cancer. Abnormalities in apoptotic function contribute to both the pathogenesis of colorectal cancer as well as its resistance to chemotherapeutic drugs and radiotherapy. An understanding of apoptosis would help enhance normal physiology and ameliorate diseases. Example of such intervention include hastening the healing or preventing chemotherapy- and radiotherapy-induced intestinal damage;to overcome apoptosis resistance in cancer cells;to retard the damage induced in inflammatory conditions such as inflammatory bowel disease, celiac disease, enteropathogenic infections and ischemia.